Involvement of the respiratory chain of gram-negative bacteria in the reduction of tellurite

The terminal oxidases of the respiratory chain of seven strains of gram-negative bacteria were shown to be involved in the reduction of tellurite. The rate of tellurite reduction correlated with the intensity of respiration. The inhibitors of terminal oxidases, carbon monoxide and cyanide, inhibited the reduction of tellurite. In Pseudomonas aeruginosa PAO ML4262 and P. aeruginosa PAO ML4262 (pBS 10), the respiratory chain was found to contain three types of cytochrome c, one of which (the carbon monoxide-binding cytochrome c) was involved in the reduction of tellurite. Agrobacterium tumefaciens VKM B-1219, P. aeruginosa IBPM B-13, and Escherichia coli G0-102bd++ cells contained oxidases aa3, bb3, and bd, respectively. The respiratory chain of other strains contained two oxidases: E. coli DH5alpha of bb3- and bd-type, and Erwinia carotovora VKM B-567 of bo3- and bd-type. All the strains under study reduced tellurite with the formation of tellurium crystallites. Depending on the position of the active center of terminal oxidases in the plasma membrane, the crystallites appeared either in the periplasmic space [P. aeruginosa PAO ML4262 and P. aeruginosa PAO ML4262 (pBS10)], or on the outer surface of the membrane (A. tumefaciens VKM B-1219 and P. aeruginosa IBPM B-13), its inner surface (E. coli G0-102bd++), or on both surfaces (E. coli DHaalpha and E. carotovora VKM B-567).     

Bacterial tellurite resistance

Tellurium compounds are used in several industrial processes, although they are relatively rare in the environment. Genes associated with tellurite resistance (TeR) are found in many pathogenic bacteria. Tellurite can be detoxified through interactions with cellular thiols, such as glutathione, or a methyltransferase-catalyzed reaction, although neither process appears involved in plasmid-mediated TeR.     

Use of diethyldithiocarbamate for quantitative determination of tellurite uptake by bacteria.

We have developed a simple method for the quantitative determination of tellurite in biological media. This assay is suitable for studying tellurite uptake in bacteria and overcomes the problems of older techniques which are time consuming and labor intensive. In earlier protocols diethyldithiocarbamate was reacted with tellurite and the resulting complex was extracted into organic solvents before spectrophotometric determination. In this study, diethyldithiocarbamate was incubated with tellurite at neutral pH to form a yellow colloidal solution. The absorbance of the aqueous yellow sol was used to determine tellurite concentrations in the range of 1 to 50 micrograms/ml (4 to 200 microM) without the need for solvent extraction.     

Glutathione is a target in tellurite toxicity and is protected by tellurite resistance determinants in Escherichia coli

Tellurite (TeO3(2-)) is highly toxic to most microorganisms. The mechanisms of toxicity or resistance are poorly understood. It has been shown that tellurite rapidly depletes the reduced thiol content within wild-type Escherichia coli. We have shown that the presence of plasmid-borne tellurite-resistance determinants protects against general thiol oxidation by tellurite. In the present study we observe that the tellurite-dependent depletion of cellular thiols in mutants of the glutathione and thioredoxin thiol:redox system was less than in wild-type cells. To identify the type of low-molecular-weight thiol compounds affected by tellurite exposure, the thiol-containing molecules were analyzed by reverse phase HPLC as their monobromobimane derivatives. Results indicated that reduced glutathione is a major initial target of tellurite reactivity within the cell. Other thiol species are also targeted by tellurite, including reduced coenzyme A. The presence of the tellurite resistance determinants kilA and ter protect against the loss of reduced glutathione by as much as 60% over a 2 h exposure. This protection of glutathione oxidation is likely key to the resistance mechanism of these determinants. Additionally, the thiol oxidation response curves were compared between selenite and tellurite. The loss of thiol compounds within the cell recovered from selenite but not to tellurite.     

Mercury resistance among clinical isolates of Escherichia coli

The use of organomercurials in liquid detergents and disinfectants promoted resistance to mercury among bacteria. Dental amalgam and industries using mercury are the main source of human exposure to mercury vapor. Release of mercury from dental amalgam contributes to the enrichment of the intestinal flora with mercury resistance plasmids which may be associated with antibiotic resistance. The aim of our study was to evaluate the frequency of E. coli strains resistant to mercury and other antimicrobial agents currently used in therapy. The bacterial mercury and ampicillin, cephalexin, cefotaxime, gentamicin, tetracycline and chloramphenicol resistance was tested against 363 E. coli strains obtained from faeces and urine between 1999-2000. According to the guidelines suggested by NCCLS (1998), minimum inhibitory concentrations (MICs) were determined on Mueller-Hinton agar, using the dilution technique with an inoculum of about 10(5) CFU. The MICs were read after 18 h incubation at 37 degrees C as the lowest concentration that inhibited the development of visible growth. Plasmids in enterobacteria may carry genes encoding resistance to both mercury and antibiotics. Among the tested E. coli strains, mercury resistance rose to 29.2%. Mercury resistance in E. coli is significantly linked to multiresistance to antimicrobial agents. Between 91.5-23.6 of mercury chloride resistant isolates were also resistant to the tested antibiotics. The increased use of non antibiotic antimicrobial agents is a possible selection factor for antibiotic-resistant strains in clinical and domestic environments.     

Impact of Manure Fertilization on the Abundance of Antibiotic-Resistant Bacteria and Frequency of Detection of Antibiotic Resistance Genes in Soil and on Vegetables at Harvest

Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.     

In vivo 31P nuclear magnetic resonance investigation of tellurite toxicity in Escherichia coli

Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by 31P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by 31P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.     

Do beta-lactam antibiotics permeabilize the outer membrane of gram-negative bacteria? An electrochemical investigation

The effects of cefotaxime and EDTA on the reducing activity of Escherichia coli and Staphylococcus aureus cultures growing in the presence of lipoic acid (LA) were investigated by potential-time measurements. The potentiometric responses of E. coli cultures exposed to EDTA indicated enhanced transmembrane transport of LA as a consequence of the outer membrane permeabilization by the chelator, whereas EDTA exerted no effect on the reducing activity of S. aureus cultures. In the same way, cefotaxime stimulated the reducing activity of E. coli, but not that of S. aureus. These results suggest that cefotaxime, and, more generally, a great variety of beta-lactam antibiotics, are able to permeabilize the outer membrane of Gram-negative bacteria.     

Comparative survival of antibiotic-resistant and -sensitive fecal indicator bacteria in estuarine water.

The survival of antibiotic-resistant and -sensitive strains of Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Streptococcus equinus, and two environmental isolates, AP17 and AQ62, was examined in estuarine water. Each strain was rendered resistant to a combination of two antibiotics by serial passage in increasing concentrations of antibiotics. Cultures were incubated in filter-sterilized estuarine water for up to 7 days. Recovery was assessed by examining colony-forming ability on media with and without antibiotics. None of the antibiotic-resistant forms survived longer than its antibiotic-sensitive counterpart in estuarine water. Three of the resistant strains died off more rapidly than the antibiotic-sensitive wild type. Survival of the test bacteria in estuarine water was as follows: sensitive and resistant AQ62, resistant Escherichia coli less than sensitive Escherichia coli less than resistant AP17 less than resistant Enterococcus faecium less than sensitive AP17, sensitive and resistant S. equinus less than sensitive and resistant Enterococcus faecalis, sensitive Enterococcus faecium. The results supported the suggestion that fecal entercocci may serve as better indicators of fecal pollution than Escherichia coli in marine ecosystems. Moreover, the results indicated that the use of antibiotic-resistant mutants to follow the fate of bacteria in the environment is inappropriate without adequate studies to ensure that resistant and wild-type strains react similarly to environmental stressors.     

Evaluation of penicillin-based inhibitors of the class A and B beta-lactamases from Bacillus anthracis

Bacillus anthracis contains a class A (Bla1) and class B (Bla2) beta-lactamase, which confer resistance to beta-lactam antibiotics when expressed in Escherichia coli. In an effort to find new beta-lactamase inhibitors, several penicillin derivatives have been evaluated including experimental compounds incorporating a 6-mercaptomethyl group or a 6-pyridylmethylidene group, along with clavulanate and tazobactam, as inhibitors against Bla1 and Bla2. The 6-mercaptomethyl-substituted penicillins showed much greater activity against the zinc-containing Bla2 than Bla1. The compound that incorporated a 6-pyridylmethylidene substituent and a catecholic substituent at the 2' position was the most effective inhibitor of Bla1 with Ki=0.057 microM. Inhibitors containing iron-chelating functional groups have previously been shown to work in combination with antibiotics to inhibit growth of antibiotic-resistant bacteria expressing beta-lactamase. The development of similar compounds, incorporating these types of substituents, may help overcome resistance to currently used antibiotics.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coli-form bacteria on three occasions during 1983. Eighty percent or more of the coli-form bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherchia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 μg/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

Isolation and initial characterization of the tellurite reducing moderately halophilic bacterium, Salinicoccus sp. strain QW6

Among the 49 strains of moderately halophilic bacteria isolated from the salty environments of Iran, a Gram-positive coccus designated as strain QW6 showed high capacity in the removal of toxic oxyanions of tellurium in a wide range of culture medium factors including pH (5.5-10.5), temperature (25-45 degrees C), various salts including NaCl, KCl, and Na(2)SO(4) (0.5-4M), selenooxyanions (2-10mM), and at different concentrations of potassium tellurite (0.5-1mM) under aerobic condition. Phenotypic characterization and phylogenetic analyses based on 16S rDNA sequence comparisons indicated that this strain was a member of the genus Salinicoccus. The maximum tellurite removal was exhibited in 1.5M NaCl at 35 degrees C, while the activity reduced by 53% and 47% at 25 and 45 degrees C, respectively. The optimum pH for removal activity was shown to be 7.5, with 90% and 83% reduced removal capacities at the two extreme values of 5.5 and 10, respectively. The impact of different concentrations of selenooxyanions (2-10mM) on tellurite removal by strain QW6 was evaluated. The ability of strain QW6 in the removal of tellurite in the presence of 6mM selenite increased by 25%. The concentration of toxic potassium tellurite in the supernatant of the bacterial culture medium decreased by 99% (from 0.5 to 0.005mM) after 6 days and the color of the medium changed to black due to the formation of less toxic elemental tellurium.     

The contribution of tellurite resistance genes to the fitness of Escherichia coli uropathogenic strains

The presence of tellurite resistance gene operons has been reported in several human pathogens despite the fact that tellurium, as well as its soluble salts, are both rare in nature and are no longer in use as antimicrobial agents. We have introduced the cloned terWZA-F genes from an uropathogenic Escherichia coli isolate into another clinical E. coli isolate that was shown to be ter-gene free. The presence of the introduced genes increased the level of potassium tellurite resistance, as well as the level of resistance to oxidative stress mediated by hydrogen peroxide; and prolonged the ability of particular strains to survive in macrophages. We therefore propose that the contribution of tellurite resistance genes to oxidative stress resistance in bacteria is at least one reason for their presence in the genomes of a broad range of pathogenic microorganisms.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

The thiol:disulfide oxidoreductase DsbB mediates the oxidizing effects of the toxic metalloid tellurite (TeO32-) on the plasma membrane redox system of the facultative phototroph Rhodobacter capsulatus

The highly toxic oxyanion tellurite (TeO3(2-)) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rhodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the QH2:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the QH2:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/pDsbB(WT), in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an "electron conduit" between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of Te(IV), the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria.     

临床分离4544株革兰阴性杆菌的耐药分布与变迁

目的了解玉溪市革兰阴性杆菌的耐药分布与变迁,为临床用药提供依据。方法对玉溪市人民医院1999—2004年临床各科送检的各类标本中培养分离出的4544株革兰阴性杆菌作回顾性分析。结果9种（属）细菌对25种药物的药敏结果耐药率〉50％者72种次（40．9％）,〈20％者44种次（25．0％）。总的耐药情况为不动杆菌和阴沟肠杆菌最高,铜绿假单胞菌和大肠埃希菌次之,甲型副伤寒沙门菌和福氏志贺菌最低。亚胺培南对多种细菌有较高的敏感覆盖率。有4种细菌对10种抗菌药物耐药率的上升具有临床意义（P〈0.01或P〈0．05）,其中以甲型副伤寒沙门菌对氟喹诺酮类上升最为显著。结论临床分离革兰阴性杆菌的耐药形势十分严峻,应定期监测区域内细菌的耐药变化．指导临床合坪用药。     

The Geobacillus stearothermophilus V iscS gene, encoding cysteine desulfurase, confers resistance to potassium tellurite in Escherichia coli K-12

Many eubacteria are resistant to the toxic oxidizing agent potassium tellurite, and tellurite resistance involves diverse biochemical mechanisms. Expression of the iscS gene from Geobacillus stearothermophilus V, which is naturally resistant to tellurite, confers tellurite resistance in Escherichia coli K-12, which is naturally sensitive to tellurite. The G. stearothermophilus iscS gene encodes a cysteine desulfurase. A site-directed mutation in iscS that prevents binding of its pyridoxal phosphate cofactor abolishes both enzyme activity and its ability to confer tellurite resistance in E. coli. Expression of the G. stearothermophilus iscS gene confers tellurite resistance in tellurite-hypersensitive E. coli iscS and sodA sodB mutants (deficient in superoxide dismutase) and complements the auxotrophic requirement of an E. coli iscS mutant for thiamine but not for nicotinic acid. These and other results support the hypothesis that the reduction of tellurite generates superoxide anions and that the primary targets of superoxide damage in E. coli are enzymes with iron-sulfur clusters.     

Alterations in growth of tissue-cultured tansy (Tanacetum vulgare L.) treated with antibiotics

Effects of three antibiotics (cefotaxime, rifampicin and gentamicin) were tested on in vitro shoots cultures of tansy (Tanacetum vulgare L.)- These antibiotics were selected because endophytic bacteria isolated from the in vitro shoot cultures of tansy showed that all bacteria were Gram-negative and their growth was reduced by these three antibiotics. Five isolates were Enterobacteriaceae, three were fluorescent Pseudomonas, and two were aerobic bacteria. Increased concentrations of antibiotics caused usually linear or quadratic changes on the initiation of shoot growth, shoot number, growth rate, and shoot height. These changes and changes in pH of the culture media were tansy genotype-dependent following treatments with gentamicin. Also, the treatment with rifampicin or cefotaxime showed a genotype-dependent effect, because they resulted in significantly higher percentage of rooted plants in one of the three tansy genotypes tested. The growth rate and length of shoots were reduced in the media containing both gentamicin and rifampicin, but less so than in media containing both gentamicin and cefotaxime.     

Activity and synergistic interactions of stilbenes and antibiotic combinations against bacteria in vitro

The synergistic antibacterial activity of two stilbenes [3,4??,5-trihydroxystilbene (1) and 3,5-dihydroxy-4-isopropylstilbene(2)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) in combination with ciprofloxacin and cefotaxime was investigated. The activity of the stilbenes and standard antibiotics on bacteria were determined using the macrodilution method. The minimum inhibitory concentration and minimum bactericidal concentration of the stilbenes was compared with that of the standard antibiotics. The antibacterial activities of stilbenes against pathogenic bacteria were assessed using the checkerboard and time-kill methods to evaluate the synergistic effects of stilbenes in treatment with ciprofloxacin or cefotaxime. The results of the present study showed that the combination effects of both stilbenes with ciprofloxacin were synergistic (FIC index <0.5). Whereas stilbene 2 with cefotaxime recorded additive. Furthermore, time-kill study showed that the growth of the tested bacteria was completely attenuated with 12?C24?h of treatment with 50:50 ratios of stilbenes and antibiotics. These results suggest that stilbenes combined with antibiotics may be microbiologically beneficial and not antagonistic. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and stilbenes). The in vitro synergistic activity of stilbenes and antibiotics against bacteria is reported here for the first time.     

Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance

The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K(2)TeO(3) at 128 microg/ml, compared to the MIC of 2 microg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a approximately 20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.